Design and implementation of degenerate microsatellite primers for the mammalian clade

Microsatellites are popular genetic markers in molecular ecology, genetic mapping and forensics. Unfortunately, despite recent advances, the isolation of de novo polymorphic microsatellite loci often requires expensive and intensive groundwork. Primers developed for a focal species are commonly tested in a related, non-focal species of interest for the amplification of orthologous polymorphic loci; when successful, this approach significantly reduces cost and time of microsatellite development. However, transferability of polymorphic microsatellite loci decreases rapidly with increasing evolutionary distance, and this approach has shown its limits. Whole genome sequences represent an under-exploited resource to develop cross-species primers for microsatellites. Here we describe a three-step method that combines a novel in silico pipeline that we use to (1) identify conserved microsatellite loci from a multiple genome alignments, (2) design degenerate primer pairs, with (3) a simple PCR protocol used to implement these primers across species. Using this approach we developed a set of primers for the mammalian clade. We found 126,306 human microsatellites conserved in mammalian aligned sequences, and isolated 5,596 loci using criteria based on wide conservation. From a random subset of ~1000 dinucleotide repeats, we designed degenerate primer pairs for 19 loci, of which five produced polymorphic fragments in up to 18 mammalian species, including the distinctly related marsupials and monotremes, groups that diverged from other mammals 120-160 million years ago. Using our method, many more cross-clade microsatellite loci can be harvested from the currently available genomic data, and this ability is set to improve exponentially as further genomes are sequenced.     

Development of microsatellite genetic markers in Siberian stone pine (<Emphasis Type="Italic">Pinus sibirica</Emphasis> Du Tour) based on the <Emphasis Type="Italic">de novo</Emphasis> whole genome sequencing

Siberian stone pine, Pinus sibirica Du Tour is one of the most economically and environmentally important forest-forming species of conifers in Russia. To study these forests a large number of highly polymorphic molecular genetic markers, such as microsatellite loci, are required. Prior to the new high-throughput next generation sequencing (NGS) methods, discovery of microsatellite loci and development of micro-satellite markers were very time consuming and laborious. The recently developed draft assembly of the Siberian stone pine genome, sequenced using the NGS methods, allowed us to identify a large number of microsatellite loci in the Siberian stone pine genome and to develop species-specific PCR primers for amplification and genotyping of 70 microsatellite loci. The primers were designed using contigs containing short simple sequence tandem repeats from the Siberian stone pine whole genome draft assembly. Based on the testing of primers for 70 microsatellite loci with tri-, tetra- or pentanucleotide repeats, 18 most promising, reliable and polymorphic loci were selected that can be used further as molecular genetic markers in population genetic studies of Siberian stone pine.     

豚鼠基因组26个多态性微卫星标记的筛选

目的筛选豚鼠基因组的多态性微卫星标记,为豚鼠遗传质量控制及基因定位等工作奠定基础。方法采用磁珠富集法和豚鼠基因组数据库筛选法获取微卫星位点序列,通过分析和初步筛选,挑选部分候选位点,根据其序列设计引物,对5种不同来源的豚鼠基因组DNA标本进行PCR扩增,以期获得多态性分子标记。结果本实验采用磁珠富集法共获得微卫星序列304个,设计引物125对,最终获得多态性位点1个,暂未发现多态性的特异性位点17个;用数据库筛选法共获得微卫星序列292个,设计并合成相应引物178对,最终发现多态性位点25个,暂未发现多态性的特异性位点28个。结论本实验获得26个多态性微卫星标记,45个潜在的候选标记,为微卫星标记在豚鼠遗传质量监测及突变基因定位等工作的应用奠定了基础。     

Characterization of microsatellite loci for the Argentine ant,Linepithema humile,and their potential for analysis of colony structure in invading Hawaiian populations

We developed primers for five polymorphic microsatellite loci to analyse the genetic structure of colonies in an invading Argentine ant population located in Haleakala National Park on the island of Maui, Hawaii. Microsatellite loci were isolated using both a polymerase chain reaction‐based and a cloning‐based method. With a range of 3–18 alleles and expected levels of heterozygosity of 0.46–0.77, these loci provide useful markers for the detection of colony and population structure in new or expanding populations of this species.     

Characterization of 24 microsatellite markers in 11 species of fire ants in the genus Solenopsis (Hymenoptera: Formicidae)

The social parasite ant Solenopsis daguerrei infests colonies of several mound-building fire ant species. Twenty-four microsatellite markers were isolated from a repeat-enriched genomic library of S. daguerrei. Eleven loci were polymorphic in this ant with two to six alleles per locus. Expected heterozygosity ranged from 0.0222 to 0.7940 among loci. Most microsatellites amplified successfully across the 11 Solenopsis species tested and will be useful for evolutionary genetic studies in this diverse ant group.     

Isolation and characterization of polymorphic microsatellite loci from the invasive plant Solidago canadensis (Asteraceae)

Solidago canadensis, a clonal herb originally from North America (common name: Canada goldenrod), is an invasive species in many countries. We developed microsatellite primers for this species. Eleven polymorphic loci were generated and primers were designed. Polymorphism of these 11 loci was assessed in 35 plants from two populations (Wuhan and Shanghai) in China. The number of alleles per locus ranged from 3 to 14. The observed and expected heterozygosities varied from 0.0732 to 0.7391 and from 0.1177 to 0.8687, respectively. These microsatellite markers will be useful tools for studies of population genetics in the native and invasive range of this species.     

Characterization and PCR multiplexing of polymorphic microsatellite loci for the invasive ant Wasmannia auropunctata

Highly polymorphic genetic markers provide a useful tool for estimating genetic parameters in studies of the evolution of sociality in insects. We isolated and characterized 12 polymorphic microsatellite loci in the invasive ant, Wasmannia auropunctata, and described experimental conditions for PCR (polymerase chain reaction) multiplexing and simultaneously genotyping these loci in two sets of five and seven markers. The number of alleles per locus ranged from two to 14 and the observed heterozygosity ranged from 0.233 to 0.967. Moreover, results of cross‐species amplification tests are reported in three other species of Wasmannia and in two species of the genus Allomerus.     

Development of 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

Microsatellite primers for fungus‐growing ants

We isolated five polymorphic microsatellite loci from a library of two thousand recombinant clones of two fungus‐growing ant species, Cyphomyrmex longiscapus and Trachymyrmex cf. zeteki. Amplification and heterozygosity were tested in five species of higher attine ants using both the newly developed primers and earlier published primers that were developed for fungus‐growing ants. A total of 20 variable microsatellite loci, developed for six different species of fungus‐growing ants, are now available for studying the population genetics and colony kin‐structure of these ants.     

Isolation and characterization of 10 new compound microsatellite markers for a mangrove tree species,Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

Avicennia marina is an ecologically important mangrove tree species. We isolated 10 polymorphic microsatellite loci from this species using an improved technique. Our isolated loci provided compound microsatellite markers with polymorphism of two to six alleles per locus. The observed and expected heterozygosities ranged from 0.025 to 0.625 and from 0.096 to 0.767, respectively. These markers would be the useful tools for researching on the genetic diversity and population genetic structure of A. marina.     

Cross-priming of microsatellite loci in subfamily cyprininae (family Cyprinidae): their utility in finding markers for population genetic analysis in three Indian major carps

This study is aimed to identify polymorphic microsatellite markers and establish their potential for population genetics studies in three carp (family cyprinidae; subfamily cyprininae) species, Labeo rohita, Catla catla and Cirrhinus mrigala through use of cyprinid primers. These species have high commercial value and knowledge of genetic variation is important for management of farmed and wild populations. We tested 108 microsatellite primers from 11 species belonging to three different cyprinid subfamilies, Cyprininae, Barbinae and Leuciscinae out of which 63 primers (58.33 %) successfully amplified orthologous loci in three focal species. Forty-two loci generated from 29 primers were polymorphic in these three carp species. Sequencing of amplified product confirmed the presence of SSRs in these 42 loci and orthologous nature of the loci. To validate potential of these 42 polymorphic loci in determining the genetic variation, we analyzed 486 samples of three focal species collected from Indus, Ganges and Brahmaputra river systems. Results indicated significant genetic variation, with mean number of alleles per locus ranging from 6.80 to 14.40 and observed heterozygosity ranging from 0.50 to 0.74 in the three focal species. Highly significant (P < 0.00001) allelic homogeneity values revealed that the identified loci can be efficiently used in population genetics analysis of these carp species. Further, thirty-two loci from 19 primers were useful for genotyping in more than one species. The data from the present study was compiled with cross-species amplification data from previous results on eight species of subfamily cyprininae to compare cross-transferability of microsatellite loci. It was revealed that out of 226 heterologous loci amplified, 152 loci that originated from 77 loci exhibited polymorphism and 45 primers were of multispecies utility, common for 2–7 species.     

Six new polymorphic microsatellite loci isolated and characterized from the African savannah elephant genome

The African savannah elephant (Loxodonta africana) is a ‘keystone’ species that plays a vital role in regulating the dynamics of both plant and animal communities and yet it is endangered and its numbers have been reduced to approximately 500 000 across their entire continental range. Molecular genetic markers are important tools for providing genetic information useful in formulating effective management and conservation strategies for the surviving elephant populations. We describe the isolation and characterization of six new polymorphic microsatellite markers in the African savannah elephant and demonstrate that these loci can be PCR (polymerase chain reaction)‐multiplexed, a desirable attribute that saves costs in large‐scale microsatellite screening.     

Isolation and characterization of microsatellite loci in Merluccius australis and cross-species amplification

Eight novel and two heterologous microsatellite pairs of primers are presented for the Austral hake (Merluccius australis), representing the first microsatellite markers available for this species. Loci were characterized for 50 individuals from two populations in South America (Argentinean and Chilean coasts). All loci were polymorphic within M. australis (5 to 30 alleles per locus; observed heterozygosity between 0.320 and 0.840), and therefore useful for population genetic studies within the species. Cross-species transferability was tested for 100 individuals from four additional species within the Merluccius genus (M. albidus, M. bilinearis, M. gayi and M. hubbsi), and results indicate that most of these primers pairs will likely be useful for population genetic studies on Merluccius species.     

Microsatellite polymorphisms in Bolivian squirrel monkeys (Saimiri boliviensis)

Two different approaches were used to identify new microsatellite polymorphisms among captive Bolivian squirrel monkeys (Saimiri boliviensis). In the first case, PCR primers for published human microsatellite loci were screened using genomic DNA from squirrel monkeys. Six polymorphic loci were identified using DNA samples from 19 unrelated individuals. The average heterozygosity among these six loci is 0.73. In the second set of experiments, a DNA library was created from Saimiri genomic DNA, and clones were selected from that library by screening with probes containing di-, tri-, and tetranucleotide repeats. Six novel microsatellites were identified this way, with an average heterozygosity of 0.59. Primer pairs for these six cloned microsatellites were also screened using a series of DNA samples from ten other platyrrhine species to assess the potential utility of these loci in other taxa. This study provides 12 new DNA polymorphisms that will be useful for various studies of this genus and demonstrates that both approaches can be used to develop new DNA polymorphisms in platyrrhine species.     

High degree of transferability of 86 newly developed zebra finch EST-linked microsatellite markers in 8 bird species

High-resolution analysis for population genetic and functional studies requires the use of large numbers of polymorphic markers. The recent increase of available genetic tools is facilitated by the use of publicly available expressed sequence tag (EST) sequence databases that are a valuable resource for identifying gene-linked markers. In the present study, we applied bioinformatics analyses to identify microsatellite markers present in EST sequences from a zebra finch (Taeniopgia guttata) EST database and we explore the success of cross-species amplification of EST-linked microsatellite markers in 7 passerine and 1 nonpasserine species. Eighty-six zebra finch EST-linked microsatellite loci were screened for polymorphism revealing a high amplification success rate and adequate levels of polymorphism (33.3-51%) for relatively closely related species, whereas success decreased in the most distantly related species to zebra finch. EST-linked microsatellites appear to be more highly transferable between taxa than anonymous microsatellites as they revealed higher amplification and polymorphism success between different families indicating that they will be a useful source of gene-linked polymorphic markers in a broad range of avian species.     

Novel polymorphic microsatellite DNA markers from Malaysian giant freshwater prawn, Macrobrachium rosenbergii

Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.     

Microsatellites in Aureliana fasciculata var. fasciculata (Solanaceae), a shrub that inhabits the Atlantic Rainforest

? Premise of the study: Microsatellite primers were developed in Aureliana fasciculata var. fasciculata, a shrub that inhabits the Atlantic Rainforest, to investigate the intraspecific genetic structure of the species. ? Methods and Results: Nine pairs of microsatellite primers were designed that were based on a simple sequence repeat (SSR)-enriched library. Seven loci were amplified successfully. Two populations of A. fasciculata var. fasciculata were genotyped and all loci were polymorphic, with two to five alleles per locus per population detected. Cross-species amplification was achieved in other members of the genus and members of the sister genus Athenaea. ? Conclusions: The development of these microsatellite markers will contribute to future population genetic studies in A. fasciculata var. fasciculata. Cross-amplification among other species in the genus and in the sister genus Athenaea might be a useful tool to establish patterns of genetic variability within the Atlantic Rainforest.     

Highly polymorphic microsatellite loci for Speyeria idalia (Lepidoptera: Nymphalidae)

Four polymorphic microsatellite loci were identified in the butterfly Speyeria idalia. We constructed a phagemid library and screened approximately 120 000 inserts. Probing with GT15, we identified 36 positives and designed polymerase chain reaction (PCR) primers for 12 potential loci. Of those loci, only four consistently produced polymorphic, diallelic PCR products in the expected size range. These results are consistent with previous studies concerning the low frequency of microsatellite loci in the lepidoptera, although these four loci are highly polymorphic and therefore likely to provide information on the fine scale genetic structure among populations in this species.     

Novel polymorphic microsatellite DNA markers from Malaysian giant freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis>

Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.